DESCRIPTION (Adapted from the applicant s abstract): M. pneumoniae is the leading cause of pneumonia in the age group that includes older children and young adults. This cell wall-less prokaryote is a member of a taxonomic class that includes numerous pathogens in humans, domestic animals, plants, and invertebrates. Despite their impact on public health and agriculture, mycoplasmas are poorly understood. Just as modern medicine is grounded in the detailed understanding of human anatomy, physiology, cell biology and gene regulation, more effective means of prevention, control and resolution of infections requires the elucidation of the basic biology and virulence factors of the organisms involved. Until recently, however, mycoplasmas have been refractory to the techniques of genetics and molecular biology that have contributed to major advances in defining the function and regulation of virulence factors in other important pathogens. Adherence of M. pneumoniae to the respiratory epithelium (cytadherence) is essential to successful colonization and disease. Cytadherence is a complex process involving adhesin proteins as well as accessory proteins required for the proper distribution and disposition of adhesins on the mycoplasma surface. The focus of this proposal is the detailed analysis of the operon that includes the genes for the adhesin, P30, and the cytadherence-accessory proteins, HMW1 and HMW3, as well as a cytadherence regulatory locus (crl) required for expression of HMW1 and HMW3. Nucleotide sequence analysis predicts that the genes for HMW1 and HMW3 are components of a multicistronic operon. The start site for transcription will be established by primer extension, and sequences required for expression will be evaluated using a reporter system and genetic complementation. Transcript levels will be compared by RNA slot blot hybridization. Transposon insertion sites in crl will be mapped, and excision deletion mutants isolated and characterized. Finally, the products of the other open reading frames in the hmw operon will be identified and their subcellular location established. Their requirement for the localization of HMW3 to, and assembly of, the attachment organelle will be assessed by immuno-electron microscopy of mutant strains.